Name: analysis of the c-kit ligand promoter using chromatin immunoprecipitation
Uploaded: 2017-06-27T15:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation at JoVE.com

这项工作得到德克萨斯大学MD安德森癌症中心，德克萨斯州休斯顿（启动基金会BB）的支持。

1.准备解决方案<ol><li> 为每个实验准备以下解决方案。 <ol><li>对于固定溶液 ，制备37％甲醛并将其储存在室温下。在1x磷酸盐缓冲盐水（PBS）中稀释至1.42％的最终浓度。 注意:甲醛被分类为刺激性，腐蚀性，致突变性，致畸性和致癌性。不要摄取不要吸入气体/烟雾/蒸气/喷雾。在通风不足的情况下，佩戴合适的呼吸设备。避免与皮肤和眼睛接触。远离不兼容的物质，如氧化剂，还原剂，酸，碱和水分。 </li><li>对于甘氨酸止痛溶液，在1×PBS溶液中制备0.125M甘氨酸并将其储存在室温下。 </li><li>对于细胞刮擦溶液，在dH 2 O中制备1×PBS溶液并将其储存在冰上。直接使用前，加入蛋白酶抑制剂苯甲磺酰氟（PMSF）t最终浓度为0.5mM。 小心:PMSF分类为腐蚀性和毒性;穿防护服，并在通风的地方使用。 </li><li>在使用细胞裂解缓冲液（20mM Tris-HCl; pH 8.0,85mM KCl和0.5％NP40）之前，直接加入1X蛋白酶抑制剂混合物（PIC; 100X储存液在二甲基亚砜（DMSO）中:104mM 4-（ 2-氨基乙基）苯磺酰氟化物盐酸盐（AEBSF），80μM抑肽酶，4mM bestatin，1.4mM E-64,2mM亮抑酶肽和1.5mM胃酶抑素A）和0.5mM PMSF。 </li><li>在使用细胞裂解缓冲液（50mM Tris-Cl; pH8.0,10mM乙二胺四乙酸（EDTA）和1％SDS）之前，直接加入1X PIC和0.5mM PMSF。 </li></ol></li></ol>细胞固定和剪切<ol><li>在10厘米的组织培养板上将细胞生长至70-80％汇合。根据实验目标刺激细胞。 </li><li>取出组织培养基，加入5 mL固定液，孵育细胞在摇床上在室温下10分钟。 </li><li>取出固定液并用10mL冰冷的1×PBS洗涤细胞两次。 </li><li>取出1×PBS，加入5 mL甘氨酸止痛溶液，并在摇床上室温孵育细胞5 min。 </li><li>取出甘氨酸止血溶液，并用10ml冰冷的1×PBS洗涤细胞两次。 </li><li>取出1×PBS，加入2mL细胞刮擦溶液，并用细胞刮刀收集细胞。将收获的细胞转移到冰上的锥形管中。 </li><li>在600×g和4℃下将样品离心10分钟。 </li><li>取出上清液，将沉淀重悬于1 mL 1X细胞裂解缓冲液中，并在冰上孵育30 min。或者，将颗粒快速冻结在液氮中并将其储存在-80℃。 </li><li>一旦将沉淀重新悬浮在细胞裂解缓冲液中，将细胞悬浮液转移到冰冷的均质器中，并使用B型瘟疫e用于细胞破坏。 </li><li>将细胞悬浮液转移至微量离心管，并以​​2,400 xg和4℃离心样品10分钟。 </li><li>弃去上清液，将沉淀物重悬于350μL核消化缓冲液中，并在37℃下孵育样品5分钟。 </li><li>加入微球菌核酸酶（1U /μL）并通过涡旋混合。在37℃下孵育样品5-20分钟;通过反演每2分钟混合样品。 注意:时间和酶浓度在细胞系之间不同，并且可能需要在次优酶剪切的情况下进行优化。 </li><li> 或者，通过超声处理使用市售的超声波器实现染色质剪切。 注意:超声波条件将需要优化。以下使用300μL的样品体积和25％的样品处理能力提供优化示例协议。 <ol><li>在步骤2.10之后，丢弃上清液并重悬在1.0mL商业剪切缓冲液中的核颗粒。 </li><li>将等分试样将300μL再悬浮的核颗粒沉积到三个1.7mL微量离心管中。将它们放在冰上。 </li><li>使用3种不同的条件，以25％功率剪切固定染色质的3个等分试样，以确定最佳超声处理条件: 每个20秒的5个脉冲，每个脉冲之间在冰上休息30秒。 10秒脉冲20秒，每个脉冲之间在冰上休息30秒。 每个20秒的20个脉冲，每个脉冲之间在冰上休息30秒。 </li><li>继续执行步骤2.15，如下所述。 </li></ol></li><li>在与微球菌核酸酶孵育后，加入7μL冰冷的0.5M EDTA终止反应。 </li><li>用剪切的DNA在16,200 xg和4℃下离心10分钟。 </li><li>将剪切的含DNA上清液转移到新鲜的微量离心管中。将等分试样放入一个单独的微量离心管中，以确认是否存在成功剪切DNA（第3节）。立即使用剩余容量进行免疫沉淀（第4部分）或将其储存在-80°C。 </li></ol> 3.确认剪切效率<ol><li>向步骤2.16的剪切染色质的50μL等分试样中加入150μL无核酸酶的dH 2 O和10μL5 M NaCl。 </li><li>在65℃下孵育样品4小时以逆转交联。 </li><li>加入2μLRNase A（10μg/μL），37℃孵育15 min。 </li><li>加入10μL蛋白酶K（0.5μg/μL），并将样品在42℃孵育90分钟。 </li><li> 为了纯化DNA，进行苯酚/氯仿萃取12 。 <ol><li>加入无核酸酶的水至最终体积为200μL。向样品中加入200μL苯酚:氯仿:异戊醇（25:24:1）并旋涡20秒。 </li><li>室内离心机以16,000 x g的速度进行5分钟。仔细取出上部水相并将其转移到新鲜管中。在移液时请确保不要携带任何苯酚。 </li><li>加入0.1体积的3M乙酸钠（NH 4 OAc），pH5.2，并用手指轻拍管子进行混合。加入2.5体积的冰冷的100％乙醇，并通过涡旋混合5秒。将样品O / N在-20℃下孵育，或在-80℃下孵育至少1小时。 </li><li>在4℃和16,000×g离心样品30分钟以沉淀cDNA。 </li><li>仔细取出上清液，不会破坏沉淀物。加入1 mL RT，70％乙醇，并倒置多次。 </li><li>在4℃和16,000×g离心样品2分钟。仔细清除上清液。 </li><li>在室温下干燥沉淀5-10分钟。将沉淀重悬于30μL的dH 2 O. </li></ol></li><li>使用5和10μL等分的纯化DNA进行凝胶电泳，并用1％TAE进行凝胶电泳琼脂糖凝胶。 注意:成功的染色质剪切导致200-1,500 bp的DNA模式。如果剪切不成功，则通过将剪切步骤（步骤2.12）的孵育时间修改为5,10和15分钟来确定特定细胞的最佳剪切条件。 </li><li>使用剩余样品分析样品的DNA浓度。反向计算剪切染色质样品中的DNA浓度（步骤2.16）;每个治疗组使用相同的DNA量进行免疫沉淀（第4部分）。 </li></ol> 4.免疫沉淀<ol><li>将10-25μg剪切的交联染色质（步骤2.16）与25μLChIP级蛋白G磁珠，10μL免疫沉淀稀释缓冲液（储备溶液:0.01％SDS，1.1％Triton X-100,16.7mM Tris-HCl; pH 8.0,1.2mM EDTA和167mM NaCl）和1μLPIC。 </li><li>孵育4小时后，加入1-10μg抗体（抗体浓缩物取决于抗体的亲和力;应首先根据制造商的建议使用并根据需要进行实验优化）至1.5 mL微量离心管，并加入dH 2 O至终体积为100μL。 注意:代替上述直接免疫沉淀法，可以通过首先将染色质与抗体结合并随后加入蛋白G磁珠来使用间接免疫沉淀法。 </li><li>在4℃下将样品在端对端旋转器上孵育4-12小时。 </li></ol>洗脱<ol><li>将管放在磁性支架上。一旦磁珠聚集在管的侧面，小心地取出并丢弃上清液。 </li><li>用800μL洗涤缓冲液1（0.1％SDS，1％Triton X-100,2mM EDTA pH8,20mM Tris-HCl; pH8和150mM NaCl）洗涤珠珠三次;通过反转管多次混合。将管放在磁性支架上a一旦磁珠集中在管的侧面，请小心地取出并丢弃洗涤缓冲液。 </li><li>用800μL洗涤缓冲液2（0.1％SDS，1％Triton X-100,2mM EDTA; pH8,20mM Tris-HCl pH8和500mM NaCl）洗涤珠子一次;通过反转管多次混合。一旦磁珠在管的侧面聚集，将管放在磁性支架上，小心地取出并丢弃洗涤缓冲液。 </li><li>将珠子悬浮在50μL洗脱缓冲液（1％SDS和100mM NaHCO 3 ）中，并将样品在端到端旋转器上在室温下孵育15分钟。 </li><li>将管放置在磁性支架上，一旦磁珠收集在管的侧面，将上清液转移到新管中。 </li><li>加入6μL5 M NaCl，反向交联，加入2μL蛋白酶K（0.5μg/μL）。混合样品后，在65℃下孵育样品2小时。 </li><li>为了纯化DNA，请执行一次nol /氯仿提取12 （步骤3.5），随后将沉淀重悬于30μL的dH 2 O. 注意:样品可以直接分析蛋白质结合位点（第5部分），或者可以保存在-20°C。 </li></ol>结合位点分析<ol><li>使用PCR或qRT-PCR对已知核酸序列内的特异性结合位点进行分析。对于引物设计，遵循常规PCR或qRT-PCR方案。设计引物以合成一个150-400bp的桥连蛋白结合位点的扩增子（ 图1 ）。 </li><li>使用四种不同的DNA模板设置PCR，以确保特异性:i）用感兴趣的抗体免疫沉淀的ChIP的DNA，ii）用非特异性IgG抗体免疫沉淀的ChIP的DNA，iii）输入DNA，和iv）H 2 O作为对PCR进行阴性对照以排除污染。在途径刺激的情况下使用针对免疫沉淀蛋白的已知结合位点特异性的不同的引物组选择第五模板。 注意:在c-KIT配体启动子分析的情况下，使用常规PCR来证明c-KIT配体启动子区域内的SBE上的TGF-β诱导的SMAD结合。作为阳性对照，对作为TGF-β诱导的SMAD结合的已知靶标的纤溶酶原激活物抑制剂-1（PAI-1）进行PCR。 </li></ol>

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作者宣称他们没有竞争的经济利益。

在本报告中，我们证明了TGF-β1诱导的SMAD2与c-KIT配体启动子内的SBE和TGF-β1诱导的STAT3与TGF-β1配体基因内识别序列的结合的结合。我们使用染色质免疫沉淀显示细胞因子诱导的两种转录因子的结合。 染色质免疫沉淀是一个有力的工具，用于证明目标蛋白与DNA的直接结合，以表征诱导蛋白质与DNA结合的刺激物，并表征蛋白质结合的DNA序列。后一信息可以帮助鉴定由特定目的蛋白?...

在真核生物核中，DNA与组蛋白和非组蛋白相互作用，并被凝聚成染色质。在生理，病理生理和细胞生物学环境中，细胞功能在空间上暂时受染色质协调基因表达控制。 DNA-蛋白质相互作用在细胞过程的调节中起重要作用，如DNA复制，重组和修复以及蛋白质表达。因此，DNA-蛋白质相互作用的分析是评估基因表达和细胞功能不可缺少的工具。 存在几种用于评估体外 DNA-蛋白质相互作用的技术 ，例如电泳（凝胶）移动性测定（EMSA）和DNA酶I印迹1,2 。然而，这些技术不分析染色质和细胞环境中的蛋白质 - DNA相互作用。 ChIP我sa技术捕获与其特异性DNA结合位点结合的蛋白质，从而有助于鉴定染色质环境中的DNA-蛋白质相互作用。该技术最初由Gimour和Lis开发，用于评估RNA聚合酶II与大肠杆菌和果蝇中的特异性基因的结合3,4 。通过固定DNA-蛋白复合物，然后进行染色质提取并将DNA剪切到约200个碱基对（bp）片段中。随后，通过免疫沉淀分离感兴趣的DNA结合蛋白。在DNA-蛋白质交联反转后，纯化和分析DNA。可以使用几种方法来分析蛋白质结合位点，并且取决于靶基因5内的蛋白质结合位点的核酸序列。在DNA序列已知的情况下，标准Pol可以使用侧翼于已知结合位点的特异性引物对应用ymerase链反应（PCR）。定量实时PCR（qRT-PCR）也可以使用6 。在序列未知的情况下，ChIP可以与DNA微阵列（片上芯片），DNA测序（ChIP-seq）或克隆技术7,8,9组合 。 TGF-β途径具有很强的肿瘤抑制功能，是细胞分化的关键途径。它通过TGF-β1配体与其同源受体复合物的结合而被激活，导致SMAD2 / 3转录因子的丝氨酸磷酸化。在与常见介体SMAD4结合后，SMAD复合物转移到细胞核并与靶基因启动子区域内的SBE结合，其中它调控控制细胞周期，细胞凋亡和细胞分化的基因上。 TGF-β刺激的转录反应是细胞类型和上下文特异性10 。最近，我们描述了TGF-β和c-KIT途径之间的正反馈回路11 。在该模型中，TGF-β1激活的SMAD2结合c-KIT配体启动子并诱导其表达和分泌。随后，c-KIT配体以自动和对位方式激活c-KIT受体。 c-KIT受体活化通过JAK1 / 2导致STAT3 Tyr 705-磷酸化。 STAT3激活和核易位后，STAT3结合TGF-β1配体基因并调节其表达。 在这里，我们证明ChIP分析对于识别SMAD2与c-KIT受体配体启动子的结合以及鉴定信号转导子（和）转录3（STAT3与TGF-β1-基因）。

<table><tbody><tr><td>HepG2 cells</td><td>ATCC</td><td>HB-8065</td><td></td></tr><tr><td>Hep3B cells</td><td>ATCC</td><td>HB-8064</td><td></td></tr><tr><td>TGF-&amp;beta;1</td><td>R&amp;amp;D Systems</td><td>101-B1</td><td>Used at a concentration of 10 ng/mL</td></tr><tr><td>Anti-SMAD2 antibody</td><td>Cell Signalling Technology</td><td>5339</td><td>Amount used/IP: 3 &amp;micro;g</td></tr><tr><td>Anti-STAT3 antibody</td><td>Cell Signalling Technology</td><td>4904</td><td>Amount used/IP: 3 &amp;micro;g</td></tr><tr><td>ChIP-IT Protein G Magnetic Beads</td><td>Active Motif</td><td>53033</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Active Motif</td><td>37490</td><td></td></tr><tr><td>Micrococcal Nuclease</td><td>Cell Signalling Technology</td><td>10011</td><td></td></tr><tr><td>PCR forward primer: PAI-1</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-GGAAGAGGATAAAGGACAAGCTG-3&amp;rsquo;</td></tr><tr><td>PCR reverse primer: PAI-1</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-TGCAGCCAGCCACGTGATTGTC-3&amp;rsquo;</td></tr><tr><td>PCR forward primer: SCF</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-CACTGATGTTAATGTTCAGC-3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>PCR reverse primer: SCF</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-GCTCTAATTTAAACCTGGAGC-3&amp;rsquo;</td></tr><tr><td>PCR forward primer: TGF-&amp;beta;1 (STB-1)</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-GAGAGAGACGTGAGTGGCATGTT-3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>PCR reverse primer: TGF-&amp;beta;1 (STB-1)</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-TAGCTTTCTCTGCCTTGGTCTCCCC-3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>PCR forward primer: TGF-&amp;beta;1 (STB-2)</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-GTACTGGGGGAGGAGCGGCATC-3&amp;rsquo;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>PCR reverse primer: TGF-&amp;beta;1 (STB-2)</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-TGCCACTGTCTGGAGAGAGGTGTGTC-3&amp;rsquo;&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

analysis of the c-kit ligand promoter using chromatin immunoprecipitation

多重细胞过程，包括DNA复制和修复，DNA重组和基因表达，需要蛋白质和DNA之间的相互作用。因此，DNA-蛋白相互作用调节多种生理，病理生理和生物功能，如细胞分化，细胞增殖，细胞周期控制，染色体稳定性，表观遗传基因调控和细胞转化。在真核细胞中，DNA与组蛋白和非组蛋白蛋白相互作用，并被浓缩成染色质。可以使用几种技术工具来分析DNA-蛋白质相互作用，例如电泳（凝胶）移动性测定（EMSA）和DNA酶I足迹。然而，这些技术在体外分析蛋白质 - DNA相互作用，而不是在细胞环境中。染色质免疫沉淀（ChIP）是一种在其特定DNA结合位点捕获蛋白质的技术，从而允许鉴定DNA-蛋白质相互作用在染色质环境中。通过固定DNA-蛋白质相互作用，然后免疫沉淀所关注的蛋白质来完成。随后，表征蛋白质结合的基因组位点。在这里，我们描述和讨论ChIP并证明其分析价值，用于鉴定转录生长因子-β（TGF-β）诱导的转录因子SMAD2与酪氨酸 - 酪氨酸激酶启动子区域中SMAD结合元件（SBE）的结合，蛋白激酶试剂盒（c-KIT）受体配体干细胞因子（SCF）。

TGF-β1配体与其同源受体复合物的结合导致SMAD2 / 3转录因子的丝氨酸磷酸化，其后与其共同介质SMAD4结合。 SMAD复合体移位到细胞核。 TGF-β可以直接通过SMAD与目标基因调节区内的SBE结合或间接通过转录激活子或阻遏物的SMAD调节表达调节基因转录，其随后调节感兴趣的基因10的表达。为了测试c-KIT配体SCF是否通过直接TGF-β1诱导的SMAD2与其启动子的结合转录调...

Watch this Scientific Journal Video about Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation at JoVE.com

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

DNA-蛋白质的相互作用对多种生物过程至关重要。在评估细胞功能时，DNA-蛋白质相互作用的分析对于了解基因调控是必不可少的。染色质免疫沉淀（ChIP）是分析体内这种相互作用的有力工具。

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

analysis-c-kit-ligand-promoter-using-chromatin

Research

JoVE Journal

Genetics

7.7K 次观看.  The University of Texas MD Anderson Cancer Center.  DNA-蛋白质的相互作用对多种生物过程至关重要。在评估细胞功能时，DNA-蛋白质相互作用的分析对于了解基因调控是必不可少的。染色质免疫沉淀（ChIP）是分析体内这种相互作用的有力工具。 

视频: Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

染色质免疫沉淀（ChIP）在小鼠T细胞系

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

科研

遗传学

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

启动子捕获高分辨率, 全基因组的启动子交互分析

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

癌症研究

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

验证组蛋白乙酰转移酶抑制剂的检测

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

研究稀缺细胞中启动子相互作用组的综合方法

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifying&#160;the reagent concentration and volumes, and swapping or eliminating steps, it accounts&#160;for minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

作者聚焦:研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程  

Gene expression is regulated by interactions of gene promoters with distal regulatory elements. Here, we descirbe how low input Capture Hi-C (liCHi-C) allows the identification of these interactions in rare cell types, which were previously unmeasurable.

研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

生物学

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

使用染色质免疫沉淀分析c-KIT配体启动子

摘要

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染色质免疫沉淀（ChIP）在小鼠T细胞系

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

启动子捕获高分辨率, 全基因组的启动子交互分析

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

验证组蛋白乙酰转移酶抑制剂的检测

研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

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